A mild TCEP-based para-azidobenzyl cleavage strategy to transform reversible cysteine thiol labelling reagents into irreversible conjugates

Article abstract of DOI:10.1039/c4cc08515a

It has recently emerged that the succinimide linkage of a maleimide thiol addition product is fragile, which is a major issue in fields where thiol functionalisation needs to be robust. Herein we deliver a strategy that generates selective cysteine thiol

Full text of DOI:10.1039/c4cc08515a

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A mild TCEP-based para-azidobenzyl cleavage strategy to transform
reversible cysteine thiol labelling reagents into irreversible conjugates†
Antoine Maruani, Shamim Alom, Pierre Canavelli, Maximilian T. W. Lee, Rachel E. Morgan, Vijay
Chudasama* and Stephen Caddick
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
cysteine bioconjugation through the use of bromomaleimides and
50 bromopyridazinediones.17 To date, our approach has provided
access to complex bioconjugates in high yields, without prior
activation of reagents with reliable, reversible conjugation.
Owing to the demand for hydrolytically stable and thiol
irreversible bioconjugates that react in a chemoselective manner,
55 we naturally sought to explore the use of reagents that would
meet these criteria. During the course of developing
bromopyridazinediones for reversible cysteine bioconjugation,
we became intrigued by the prospect of pyridazinediones (PDs)
as irreversible cysteine functionalisation reagents. Previously we
60 have shown that if one of the nitrogen atoms on the PD core is
unsubstituted that the molecule does not react with thiols at
physiological pH or higher.17a We postulate that this is a
consequence of such a structure existing as its enol tautomer,
which is likely to be significantly deprotonated under
65 physiological pH (or higher), based on the reported pKa of
1-methyl-3,6-(1H,2H)-pyridazinedione being ~5.7 in H2O and the
calculated pKa of its thioether analogue,‡ 1-methyl-4-
(methylthio)-3,6-(1H,2H)-pyridazinedione, being ~5.9.18 Thiol
reactivity will therefore be greatly reduced as the electrophilicity
70 of the resulting PD-core moiety will be tuned down considerably.
As such, we set about developing a strategy where we could
generate a mono-alkylated-PD species post-bioconjugation to a
cysteine thiol to afford a thiol stable construct (see Figure 1).
It has recently emerged that the succinimide linkage of a
maleimide thiol addition product is fragile, which is a major
issue in fields where thiol functionalisation needs to be robust.
10 Herein we deliver a strategy that generates selective cysteine
thiol labelling reagents, which are stable to hydrolysis and
thiol exchange.
Advances in protein modification by chemical means have led to
the development of
a range of protein bioconjugation
15 methodologies.1 These methodologies have been successfully
applied to a number of fields such as the fluorescent tagging of
proteins,2 and the development of therapeutic protein
conjugates3,4 to treat indications such as HIV,5 cancer,6 and
malaria.7 Chemically modified proteins are also utilised as
20 diagnostics.8
The use of synthetic methodology to modify proteins has to
overcome many major obstacles, the most significant of which is
the need for high selectivity, i.e. modifying only one amino acid
type by discriminating against the other natural amino acids in a
25 protein.9 As free cysteines are extremely rare in proteins10 and the
thiol side chain has the highest nucleophilicity of all
proteinogenic groups at physiological conditions,11 it is a very
popular target for the selective and site-specific modification of
proteins.12 Moreover, with the possibility of facile cysteine
30 introduction by site-directed mutagenesis, cysteine modification
is a leading approach. The most popular strategy for labelling the
thiol moiety of cysteine residues is by alkylation with maleimides
to form thioether-succinimides.12,13 However, it has recently
come to light that such an appendage is sub-optimal owing to
35 issues of hydrolysis, and thiol exchange with reactive thiols in the
blood (e.g. albumin).14 This has major implications for biologics
that employ a maleimide motif to functionalise a protein thiol for
in vivo applications. For example, in antibody-drug conjugates
(ADCs), where an antibody delivers a toxic payload to cancerous
40 tissue selectively, the use of maleimides to attach cytotoxic drugs
to an antibody is not ideal as thiol exchange onto human serum
albumin in the blood results in off-site toxicity.14 Although recent
advances have been made in this area through the use of
hydrolysed maleimides and succinimides,14,15 a strong drive to
45 develop novel reagents for reliable, chemoselective, stable and
irreversible thiol labelling remains, and particularly for the
construction of ADCs.16
75
Fig. 1 A novel strategy for developing thiol-stable pyridazinedione
bioconjugates.
Our study began with the reaction of model protein GFP-
S147C 1 with pyridazinediones 2 and 3 to confirm our previous
Recently, we have described a novel, reversible approach to
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observations when using protein Grb2-L111C (see Figure 2).17a
These results were consistent with our previous work and
with 15 equivalents of 1-hexanethiol in THF/PBS buffer (pH 7.4)
only led to thiol exchange in the case of derivative 6. This
confirmed that a mono-alkylated-PD is unreactive to thiol (or 35 provided encouragement for our hypothesis of a mono-alkylated-
other nucleophilic functional groups on amino acid side-chains).
PD being thiol unreactive under physiological pH or higher.
5
Fig. 2 Incubation of GFPS147C 1 with pyridazinediones 2 and 3.
These initial studies paved the way for us to appraise the use of
a novel strategy for developing thiol-stable pyridazinedione
bioconjugates (see Figure 1). To do so, we needed to develop a
10 selective method for cleavage of R2 from the PD core. There are
many strategies that could be applied, however, at this juncture
we took the opportunity to develop a novel, mild and simple
method based on an azide trigger. Our desire to use an azide-
based cleavable handle originates from the bioorthogonality of
15 the azide functional group. Taking inspiration from the well-
documented work on p-aminobenzyloxycarbonyl (PABC)
linkers,19 we set about using a p-azidobenzyl cleavage strategy
(see Figure 3).
Fig. 4 Use of TCEP in our p-azidobenzyl cleavage strategy to generate
pyridazinedione derivative 7 from derivative 6, and the thiol stability of
each construct.
40
Following these encouraging results on a small molecule
study, we appraised our strategies on a model protein with a
single cysteine mutation, GFPS147C 1. Initially, GFPS147C 1
was incubated with mono-bromo-PD 5 in sodium phosphate
45 buffer (pH 8.0) for 1 h at 37 °C. As expected, this proceeded with
complete conversion and afforded GFP-derivative 8. We next
applied our TCEP cleavage strategy, by incubation of this
derivative with 10 equivalents of TCEP in phosphate buffer at pH
8.0. Satisfyingly, clean conversion to bioconjugate 9 was
50 observed, which is consistent with our small molecule study. It is
also noteworthy that no hydrolysis occurred under these
conditions, which is consistent with our previous observations on
the PD core being hydrolytically stable.17a Having established,
using mass spectrometry, that our cleavage strategy is applicable
55 on a protein, we next compared the thiol stability of 8 and 9 by
incubation with glutathione (0.5 mM) for 72 h at pH 7.4 and
37 °C. Gratifyingly, GFP-derivative 9 was completely stable
under the reaction conditions, whereas derivative 8 showed
complete thiol exchange with glutathione. This therefore
60 established proof of concept for both our strategies on a model
protein scaffold. Moreover, this work also highlights the
versatility of the PD platform with a facile shift from reversible to
irreversible constructs achieved under mild conditions.
20 Fig. 3 Use of a p-azidobenzyl cleavage strategy to generate a thiol-stable
pyridazinedione bioconjugate.
We initially evaluated our p-azidobenzyl cleavage strategy in a
small molecule study through the use of cysteine derivative 6,
formed by reaction of N-(tert-butoxycarbonyl)-L-cysteine methyl
25 ester and mono-bromo PD 5 (see ESI† for details on synthesis).
The use of an alkyne handle, which would conceptually be
retained post p-azidobenzyl cleavage, would allow for the
resulting construct to be readily functionalised by a Cu(I)-
catalyzed Azide-Alkyne Cycloaddition (CuAAC). To our delight,
30 treatment of derivative 6 with TCEP led to clean conversion to
derivative 7, thus providing proof of concept for our novel
cleavage strategy. Moreover, incubation of derivatives 6 and 7
2
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300
200
100
0
29800
a)
c)
Expected Mass: 29,801 Da
Observed Mass: 29,800 Da
25000 26000 27000 28000 29000 30000 31000 32000 33000 34000 35000
Mass, Da
30204
d) 150
Expected Mass: 30,206 Da
Observed Mass: 30,204 Da
100
50
0
30315
25000 26000 27000 28000 29000 30000 31000 32000 33000 34000 35000
Mass, Da
Fig. 6 a) The use of ‘click’ chemistry to functionalise bioconjugate 9, and
deconvoluted MS data for b) bioconjugate 10a, c) bioconjugate 10b and
d) bioconjugate 10c.
Conclusions
20 In conclusion, we have developed, via a novel p-azidobenzyl
29613
cleavage strategy, a route to thiol stable cysteine-bioconjugates
b)
c)
200
100
0
that has
a clear advantage over conventional maleimide
Expected Mass: 29,612 Da
Observed Mass: 29,613 Da
chemistry. The strategy has been demonstrated on both a small
molecule system and on a model protein, GFPS147C. Owing to
25 the plethora of fields where thiol functionalisation needs to be
robust and irreversible, e.g. in antibody-drug conjugates (ADCs),
imaging and theranostics, we believe this work will find use in a
variety of domains. We hope to deliver on the application of our
platform in a range of contexts, including ADCs, in the near
30 future.
25000 26000 27000 28000 29000 30000 31000 32000 33000 34000 35000
Mass, Da
29484
300
200
100
0
Expected Mass: 29,482 Da
Observed Mass: 29,484 Da
25000 26000 27000 28000 29000 30000 31000 32000 33000 34000
Mass, Da
Fig. 5 a) Translation of our thiol cleavage and thiol stability strategies
onto protein bioconjugates 8 and 9, and deconvoluted MS data for b)
bioconjugate 8 and c) bioconjugate 9.
Acknowledgements
The authors gratefully acknowledge the EPSRC, Ramsay
Memorial Trust and UCL for support of our programme.
Following our work on developing a novel p-azidobenzyl
cleavage strategy and obtaining a thiol stabile construct, we set
about functionalising protein scaffold 9 by the use of ‘click’
chemistry. If successful, this would result in a facile method for
5
Notes and references
functionalising the thiol-stable bioconjugate. A number of ‘click’ 35 Department of Chemistry, University College London, 20 Gordon Street,
London, WC1H OAJ, UK. Tel: +44 (0)20 7679 2077; E-mail:
v.chudasama@ucl.ac.uk
† Electronic Supplementary Information (ESI) available: [LC-MS, ES-
conditions were trialled using benzyl azide as our model azide.
10 The most promising conditions were the use of Cu(I)Br as copper
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conversion of starting material alkyne 9 to triazole bioconjugate
10a. Moreover, these conditions also allowed for clean reaction
of the alkyne derivative with a dansyl azide and a sulfo-cyanine5
15 azide to afford 10b and 10c, respectively.
MS and deconvoluted spectra for all reactions with proteins described
1
40 herein, and H and 13C NMR spectra for all small molecule constructs].
See DOI: 10.1039/b000000x/
‡ MarvinSketch and its calculator plugins were used for pKa prediction,
MarvinSketch 14.11.3.0, ChemAxon (http://www.chemaxon.com).
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